title: Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent.
abstract: 2-DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2-D gels. To precisely locate spots of immunoreactive proteins in 2-D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer-based reagent (N-102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC-MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.
authors.affiliation: Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Japan. kawasaki@yokohama_cu.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Akiko
authors.initials: A
authors.lastname: Okayama
authors.forename: Yuko
authors.initials: Y
authors.lastname: Iwafune
authors.forename: Shota
authors.initials: S
authors.lastname: Yahagi
authors.forename: Noriaki
authors.initials: N
authors.lastname: Arakawa
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D012275:Oryza / Q000737:chemistry; D010051:Ovarian Neoplasms; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D005465:Fluorocarbon Polymers
chemical_list: D007202:Indicators and Reagents; D008567:Membranes, Artificial; D019000:Phosphotyrosine; D010940:Plant Proteins; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers